The overall goal of this proposal is to obtain an in-depth understanding of the mechanistic links between alterations of the dystrophin glycoprotein complex (DGC) and impairment of the excitation-contraction coupling (ECC) process in mammalian skeletal muscle. This functional characterization will be achieved in muscle fibers from various animal models of human muscular dystrophies. We have found that Ca2+ release evoked by action potentials (APs) (or voltage-clamp pulses) in muscle fibers from two of such models, the adult mdx mouse and the phenotypic sarcospan (SSPN) overexpressing mouse (SSPN-Tg), is significantly smaller than in wild type fibers. We hypothesize that disruption of the DGC undermines the structural and functional support for the transverse tubular system (TTS) and the sarcoplasmic reticulum (SR), thus attenuating the ECC process. We will first investigate the mechanisms responsible for the impairment of Ca2+ release in mdx mice (Aim 1), the most prevalently used animal model for Duchenne Muscular Dystrophy (DMD), which lacks dystrophin in the DGC. However, since the phenotypic alterations in mdx mice are relatively benign, possibly due to utrophin substitution in the DGC, experiments will be also carried out in double knockout mdx/utrophin (mdx/utr-/-) mice that display a phenotype more comparable to that in DMD patients (Aim 2). To further characterize the link between the DGC integrity and a fully functional ECC, we will take advantage of our ability to express DGC proteins by in vivo electroporation and use transgenic animal models with other genetic conditions altering the DGC (e.g. SSPN-Tg, and Utr-TET). The last goal of the proposal is to investigate, using 2-photon laser scanning microscopy (TPLSM) the subcellular distribution of representative DGC protein components in order to assess if they are associated exclusively with the sarcolemma or if they have a more ubiquitous distribution in association with the Z-line and the TTS. This characterization will help us understand the function of the DGC in terms of ECC and sarcolemmal integrity (Aim 3). These investigations will be carried out by using electrophysiological and state-of-the-art optical methods, such as Fster resonance energy transfer (FRET) and total internal reflection fluorescence microscopy (TIRFM), to also assess the nanoscale localization of DGC and ECC proteins with respect to the internal and external leaflets of the surface and TTS membranes.